The invention relates to methods for determining values relating to the reserve bilirubin-binding capacity of serum albumin containing aqueous specimens, and to compositions for use in such methods.
It is known that serum albumin has two high-affinity bilirubin-binding sites. For the first specific bilirubin-binding site the association constant for the reversible binding of bilirubin, K.sub.assoc., is of the order of 10.sup.8 liters/mole. For the second specific bilirubin-binding site, the association constant K.sub.assoc. is of the order of 10.sup.6 liters/mole.
It is of interest to the clinician to determine a value for the reserve bilirubin-binding capacity that represents the proportion of the said first specific high-affinity bilirubin-binding sites that are available for binding bilirubin. This is especially important in the neonatal care of jaundiced newborns.
By "reserve bilirubin-binding capacity" is meant the reserve binding-capacity of the first specific high affinity bilirubin site of serum albumin, the maximum value of this capacity being 1. The blood of the jaundiced newborn contains excessively high levels of bilirubin. When the reserve bilirubin-binding capacity of the serum albumin is low, bilirubin may find its way into the brain of the new born and cause permanent damage which may required institutional care of the patient for the rest of his life. The only method which is available for avoiding this condition is blood transfusion, and such transfusions have to be made well before the reserve bilirubin-binding capacity becomes depleted, to avoid the possibility of permanent brain damage. At present, there is no satisfactory method for determining the reserve bilirubin-binding capacity. A satisfactory assay method would therefore be highly desirable and of important assistance to the care of new borns and especially in indicating when a blood transfusion is called for.
Known methods for determining the reserve bilirubin-binding capacity, such as the peroxidase technique, electrophoretic methods, Sephadex filtration, fluorescent techniques, and methods using dyes to duplicate the binding of bilirubin to albumin, are subject to various disadvantages. For instance, some of these methods require too large an amount of blood for each test, and so they cannot be used for following the rise and fall of the binding capacity since repeated tests would demand the withdrawal of an excessive quantity of blood from the neonate body. The methods that rely on dyes and on observing colorimetric changes cannot be applied to hemolysed blood samples, as the resultant coloration obscures the color changes that are to be observed.